1. Field of the Invention
This invention relates to the generation and detection of electromagnetic emissions in a multitude of samples retained in individual wells of multi-well plates or two-dimensional sample arrays in general. This invention is of particular interest in the performance of the polymerase chain reaction (PCR) with real-time detection, i.e., PCR processes in which the amounts of nucleic acid in individual reaction wells are quantitated as the PCR reaction is progressing. The invention has general applicability however to any system that simultaneously detects emission signals from a multitude of wells, receptacles, or sample spots in a two-dimensional rectangular array.
2. Description of the Prior Art
Real-time PCR is one of many examples of chemical processes that employ area imaging to monitor the progress of a multitude of reactions that are being performed simultaneously. Area imaging is the formation of an image of the emissions from a two-dimensional array of reaction wells such as the arrays used in PCR systems. The emissions are fluorescence emissions generated by fluorescent molecular groups covalently attached to the nucleic acid segments in the wells. The imaging method involves the illumination of the entire array of reaction wells to stimulate the fluorescent groups, and the detection of the radiations that the fluorescent groups emit as a result of the stimulation. The emissions are detected in the form of a two-dimensional image corresponding to the well array. The most common array is one that contains 96 wells in a single plate, the wells arranged in 12 rows of 8 wells each, but other arrays and numbers of wells are used as well. From the magnitudes of the emissions, the system determines the amount of binding, the tag concentration, and the characteristics of the fluorescent molecule in each well. This information is processed to indicate the progress of the reaction in each well. The illumination band can be narrow such as that obtained with a laser, or broad such as that obtained with a whitelight source.
Whitelight sources are an inexpensive means of providing a wide range of stimulation wavelengths. A commonly used whitelight source is a halogen bulb with a reflector. The typical halogen bulb produces light in a cone angle that ranges from 5° to 5°. The angle is controlled in part by the shape of the reflector. An example of this type of bulb is the EXN 50 MR16/40 bulb from General Electric. This bulb has a multi-faceted elliptical reflector that removes very high spatial frequencies of light (i.e., bright spots) and re-directs the dominant beam. The far-field projected light pattern resulting from a multifaceted reflector has fewer bright spots (or “hot spots”) than the far-field projected light pattern resulting from a smooth-surface elliptical reflector of the same major diameter and focal points. There is a fundamental trade-off between product size, detection efficiency filter performance and illumination cone angle.
Despite the inclusion of facets in the bulb reflector, the light from the bulb must be redirected to a certain degree in order to uniformly illuminate a two-dimensional planar array of wells. Redirection is achieved by a series of mirror surfaces positioned in the path between the bulb and the well plate. An example of an illumination system 11 of the prior art that includes redirecting mirrors is shown in FIG. 1. In this system, the well plate 12 is shown at the bottom and the whitelight source bulb 13 is positioned above and to one side of the well plate 12. Between the bulb 13 and the well plate 12 are two sets of mirrors 14, 15, the first mirror 14 having two segments 16, 17 in a wide V-shaped profile, and the second mirror 15 having three segments 18, 19, 20, the outer segments 18, 20 of which are angled slightly inward toward the inner segment 19. In both mirror sets 14, 15, the angles between the planes of adjacent segments are very small, typically less than 10°. Light reflected from both mirror sets is received by a third mirror 21 (of which only the rear surface is visible) which is a planar mirror directing the light downward toward the well plate 12. Since the light rays from the bulb 13 and hence those reflected by the mirrors are generally divergent, the light reflected by the third mirror 21 is passed through a lens 22 to render the rays parallel, or at least approximately so, before they strike the well plate. The mirror 14 with the two segments in the V-shaped profile directs reflected light to one side of the well array, specifically the side on the right in the view shown in FIG. 1, with the two segments directing the light to the upper and lower halves, respectively, of the right side of the array. The three-segment mirror 15 directs reflected light to the other side of the well array, i.e., the side on the left in the view shown in FIG. 1, with the three segments directing light to the center region of that side. Collectively, the five mirror segments are intended to distribute the light uniformly, or approximately so, across the entire rectangular well array.
Although uniform light distribution across the well array is the goal, this goal is not fully achieved because the line at the angle between adjacent segments on each mirror produces a bright line in the reflected light, i.e., a line that is brighter than regions above and below it. Accordingly, isolated rows in the well plate are illuminated and hence stimulated at a greater intensity than adjacent rows. In the prior art, this undesired difference in brightness is eliminated by masking the line, for example by applying black ink, such as from a felt-tip pen, along the line. This wastes light energy by reducing the amount of stimulation light that reaches the well plate, and is a crude method of correcting nonuniformities.
As for the detection components, various methodologies can be used, but a detection component that is typically used is a charge-coupled device (CCD) in conjunction with an appropriate lens or lens system. A series of filters is included to control both the stimulation light and the emission light to narrow bands of frequencies. One artifact of the detection system is an intensity roll-off with the angle from the center line of the optical path, i.e., a gradient along the radial direction in the detected intensities of the emissions. As a result, emissions from wells located successively further from the center are read at successively lower intensities, even if the emissions themselves are uniform across the array. This gradient is demonstrated in FIG. 2, which is a representation of an image from a 12×8 well array. The concentric circles 31, 32, 33, 34 whose centers are coincident with the center 35 of the well array represent lines of constant emission detection intensity, each circle having a detection intensity that is less than the intensity of the adjacent circle of smaller radius. The intensity thus decreases gradually as the circle radius increases.